Team:FAU Erlangen/Tour42
Generating fluorescent yeast strains
During the course of our project we generated several yeast strains differing in the way they express YFP. Originally derived from K699, the first strain created was YCYFP, which is K699 transformed with an YCplac22 with the YFP expression construct. After we had confirmed YFP fluorescence, we created a strain with the YFP construct inserted into the genome by homologous recombination with YIplac204. In this strain we tested the SCATTER constructs by co-transforming YEplac181 containing the deacetylase part and K801000 containing the TAL domains. These strains were named RT1, RT2, and RT3 (for Rpd3 and TALE1/2/3).
YFP detection
To test the regulatory constructs we wanted to create a reporter that could be easily confirmed after transforming the yeast cells. Therefore the choice fell on a fluorescent protein that we wanted to screen for via fluorescent microscopy. In the end, due to the lack of YFP filters for fluorescence microscopy in our lab, we asked another group for help who happened to have a confocal laser scanning microscope that we could use. Using the CLSM with an appropriate YFP filter we were able to confirm that the cells expressed YFP and thus continue to the second step of downregulating the expression.
While the optical confirmation of YFP fluorescence was a good start, we wanted to quantify the expression levels before and after transformation with the regulatory constructs. To do this, we chose RT-qPCR. By quantifying the level of YFP mRNA in the cells it is possible to directly derive the level of transcriptional activity.
We used Tub1, a housekeeping gene in yeast to standardize the data. As the YFP construct we used had no intron, it was necessary to run a –RT control as well, in order to find out how much genomic DNA remained in the samples during RNA preparation and to compensate for the additional material amplified in the reaction. The data shows a clear decrease in mRNA levels in the yeast strains transformed with the regulatory constructs.